Anti-frα protein monoclonal antibodies, methods of making and uses thereof

Abstract

This invention relates to a monoclonal antibody capable of recognizing human FRα antigen, its preparation method, and its use in immunoassay. The invention provides a rabbit monoclonal antibody against FRα protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2. This antibody exhibits high specificity and sensitivity, can specifically recognize cells expressing FRα protein, and is suitable for immunological assays, particularly immunohistochemical assays.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to an anti-FRα protein monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Folate receptors (FRs) are common tumor-targeting receptors belonging to the glycoprotein family. They have a molecular weight of 35-40 kDa and four subtypes: FRα, FRβ, FRγ, and FRδ. FRα, also known as FOLR1 or folate-binding protein, is a glycoprotein anchored to the cell membrane via glycosylated phosphatidylinositol (GPI). It has a high affinity for folic acid (vitamin B9) and can transport folic acid through receptor-mediated endocytosis, thereby maintaining normal cell growth and function.

[0003] Although FRα expression levels are very low in most tissues, it is expressed in a certain proportion of general epithelial cells, such as those of the apical bronchial epithelium of the lung. However, FRα is expressed at high levels in various cancers to meet the folate requirements of rapid cell division under low folate conditions. Numerous studies have found widespread high expression of FRα in epithelial tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), and non-small cell lung cancer (14-74%). FRα has been proven to be an ideal tumor marker and can serve as an important clinical indicator for tumor diagnosis and treatment. Epithelial ovarian cancer (EOC) is a highly heterogeneous group of malignant tumors, accounting for approximately 90% of ovarian malignancies. 75% of EOCs are serous ovarian cancer (SOC), while mucinous ovarian cancer (MOC) accounts for only 3%-5%. FRα is overexpressed in SOCs at a rate as high as 80%-96% and is not expressed in MOCs. Therefore, FRα is a potential biomarker and therapeutic target for serous ovarian cancer and can help differentiate between SOC and MOC. Summary of the Invention

[0004] The inventors provide a monoclonal antibody against FRα protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.

[0005] Furthermore, the monoclonal antibody specifically recognizes the FRα protein.

[0006] Furthermore, the monoclonal antibody is a rabbit monoclonal antibody.

[0007] Furthermore, the clone number of the rabbit monoclonal antibody is MXR066.

[0008] The inventors also provide a method for preparing an anti-FRα protein monoclonal antibody, wherein the antigen used to immunize rabbits is a recombinant protein, which is recombinantly expressed by Escherichia coli.

[0009] Furthermore, the recombinant protein comprises an FRα protein fragment and a HIS protein tag.

[0010] Furthermore, the FRα protein fragment has the amino acid sequence shown in SEQ ID NO.3.

[0011] Furthermore, the plasmid vector used in the recombination process was PCMV3.

[0012] The inventors also provide an immunoassay reagent for FRα protein, wherein the immunoassay reagent contains the above-mentioned monoclonal antibody against FRα protein as its active ingredient.

[0013] Unlike existing technologies, the beneficial technical effects of this invention are as follows: The above technical solution provides a rabbit monoclonal antibody against FRα protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2. This antibody has high specificity and sensitivity, can specifically recognize cells expressing FRα protein, and is suitable for immunological detection, especially immunohistochemical detection. Attached Figure Description

[0014] Figure 1 Comparison of immunohistochemical staining results of one case of ovarian serous carcinoma (left: rabbit monoclonal antibody FRα of this invention, right: commercially available FRα).

[0015] Figure 2 Comparison of immunohistochemical staining results of fallopian tube tissue (left: rabbit monoclonal antibody FRα of this invention, right: commercially available FRα). Detailed Implementation

[0016] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0017] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0018] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0019] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.

[0020] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.

[0021] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0022] In this application, expressions such as "greater than", "less than", and "exceeding" are understood to exclude the stated number; expressions such as "above", "below", and "within" are understood to include the stated number. Furthermore, in the description of the embodiments of this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times", unless otherwise explicitly specified.

[0023] Example 1 Preparation of recombinant FRα protein fragment

[0024] I. Gene Optimization and Synthesis

[0025] According to the protein sequence accession number NP_000793.1 in the NCBI database, FRα selected the protein fragment at positions 26-257, directly optimized it into a gene fragment suitable for expression in E. coli Rosetta (DE3), and cloned it into the PCMV3 vector.

[0026] The expression vector was transformed into competent E. coli Rosetta (DE3) cells. Clones were picked from the plates and inoculated for colony PCR identification. Clones with positive PCR results were selected for sequencing analysis, and clones with completely correct sequences were used.

[0027] Selecting different antigens for immunization may produce antibodies with different binding properties. This molecule also exhibits multiple variants due to alternative splicing, ultimately leading to different antibodies recognizing cells expressing the antigen in different ways. The FRα molecule was analyzed according to its published sequence. Based on its structure, antigenicity, hydrophilicity / hydrophobicity of its constituent amino acids, and secondary structure, a suitable region with good immunogenicity and soluble expression was selected for recombinant expression. The gene sequence of FRα from positions 26-257 was selected for codon optimization, resulting in a molecular weight of approximately 25 kDa. The FRα protein was obtained through sequence optimization and design using a prokaryotic expression gene sequence. The recombinant immunogen consists of an antigenic FRα protein fragment and a protein tag for recombinant protein purification; the protein tag is HIS.

[0028] II. Protein Expression and Purification

[0029] Overnight single-colony cultures were transferred to 100 mL LB medium at a ratio of 1:100. Ampicillin was added to a final concentration of 100 μg / mL, and the culture was incubated at 37°C with shaking until the OD600 reached 0.6-0.8. Then, 0.8 mmol / L IPTG was added, and the culture was incubated at 37°C with shaking for 4 hours. After harvesting, the culture was sonicated. The recombinant protein contained a histidine tag, and affinity purification was performed using a nickel column. Elution with 150 mmol / L imidazole was performed, followed by SDS-PAGE analysis. PCMV3-FRα (26-257 aa) expression was observed in the supernatant, resulting in a purified protein concentration of 1 mg / mL and a purity of 85%, meeting the requirements for animal immunization, antibody screening, and identification.

[0030] Example 2: Single B-cell sorting of rabbits producing anti-human FRα monoclonal antibodies

[0031] I. Immunoassay and ELISA Detection

[0032] The recombinant FRα protein from Example 1 was emulsified with Freund's complete adjuvant, and six rabbits were selected for immunization at a dose of 300 µg / rabbit. Booster immunizations were performed on days 21, 28, and 49, with the antigen emulsified using Freund's incomplete adjuvant at a dose of 150 µg / rabbit. Serum ELISA titers were measured after the third immunization. Rabbit A showed an immunohistochemical titer greater than 1:2.56K, indicating that the immunization effect met requirements. Serum from four rabbits that received the fourth immunization was analyzed using IHC. Based on the results (i.e., using a positive pair photograph corresponding to the antibody, where immunohistochemical staining was observed at the corresponding detection site in the positive pair photograph), one rabbit (number: A) was selected for subsequent monoclonal antibody screening.

[0033] II. Splenic cell isolation and B lymphocyte sorting

[0034] Monoclonal antibody preparation was performed on selected rabbits. Three days after final immunization, the spleen was harvested and placed in RMPI basal medium containing 100 U / mL penicillin and 100 μg / mL streptomycin. The spleen was diced into small pieces with a scalpel and then transferred to a 100 µm cell sieve for grinding. The resulting cell suspension was filtered to remove large cell clumps and tissue membranes. After centrifugation at 400g for 5 minutes, the supernatant was discarded, and the spleen cell clumps were retained. The spleen cell clumps were resuspended in hypotonic solution, and erythrocytes were lysed. The cells were then centrifuged again at 400g for 5 minutes, retaining the spleen cells. The spleen cells were resuspended in RMPI basal medium containing 100 U / mL penicillin and 100 µg / mL streptomycin, centrifuged at 400g for 5 minutes, and then resuspended in complete medium (RMPI basal medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin) for later use.

[0035] For specific steps of B lymphocyte sorting, please refer to Chinese Patent 201910125091.4, "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".

[0036] Approximately 2000 single B cell clones were sorted and cultured. Positive clones that specifically recognized the recombinant FRα protein in Example 1 were initially screened by ELISA. Based on the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0037] III. Detection of positive clones from B cell culture

[0038] IHC validation was performed on single B cell culture supernatants using multi-tumor tissue microarrays containing FRα-positive and negative tumors and normal tissue microarrays. IHC results were obtained from the culture supernatants of 8 clones. LEM supernatants were prepared and IHC validation was performed to identify clones with excellent sensitivity and specificity (MXR066).

[0039] The sensitivity and specificity screening criteria were as follows: Immunohistochemical staining was observed at the corresponding detection sites in the positive pair photographs corresponding to the antibody, while no immunohistochemical staining was observed at the non-detection sites. The staining intensity was equal to or even higher than that of the control antibody.

[0040] IV. Cloning of the rabbit monoclonal antibody gene (MXR066) and construction of the rabbit monoclonal antibody expression plasmid

[0041] Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. Using PCR, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clones and sequenced. Rabbit monoclonal antibody expression vector plasmids were constructed using the aforementioned naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences.

[0042] Example 2 was commissioned by Fuzhou Maixin Biotechnology Development Co., Ltd. to Dima Biotechnology (Wuhan) Co., Ltd., with Fuzhou Maixin Biotechnology Development Co., Ltd. performing immunohistochemical screening.

[0043] Example 3: Expression of FRα rabbit monoclonal antibody

[0044] I. Plasmid Amplification and Extraction

[0045] Take out a tube (100 μL) of competent bacteria (DH5α), insert it onto ice, and incubate on ice for 5-10 min; add 5 μL of plasmid, gently shake, and place on ice for 30 min; gently shake well and place in a 42℃ water bath for 90 s for heat shock, then quickly return to ice and let stand for 5 min; in a clean bench, add 800 μL of LB medium (note: antibiotic-free) to the above mixture, gently mix, and fix on a shaker at 37℃ for 1 h; in a clean bench, take 50-100 μL of the above transformation mixture and drop it onto labeled solid LB agar plates containing Amp, and spread evenly with a sterile glass spreader; first, incubate upright in a 37℃ incubator for 30 min to allow the bacterial solution on the surface to completely penetrate into the medium, then invert and incubate overnight in a 37℃ incubator. Pick a single colony with a pipette tip and inject it into 4 mL of LB medium (containing 2 uL of 200 mg / mL LAmp). Incubate at 37°C for 16 h on a shaker at 220 rpm.

[0046] Add 1 mL of bacterial culture to 100 mL of LB medium (containing 50 μL of 200 mg / mL Amp) and incubate at 37 °C in a shaker for 16 h; use the SanPrep endotoxin-free material DNA mini-extraction kit (Sangon Biotech) to extract plasmids according to the instructions.

[0047] II. Transfection

[0048] The concentration of 293F cells was adjusted to 2.5–3 × 10⁻⁶ using Expi293F™ Expression Medium. 6 Viable cells / mL, and cultured overnight.

[0049] 1) Using a hemocytometer, the viable cell concentration is approximately 4.5-5.5 × 10⁻⁶. 6 The number of viable cells / mL must meet the requirements of the expression system.

[0050] 2) Dilute the cell concentration to 3×10⁻⁶ using Expi293FTM Expression Medium. 6 viable cells / mL;

[0051] 3) Add plasmid DNA to Opti-MENTM I Ruduced Serum Medium, gently pipette and invert to mix;

[0052] 4) Gently invert ExpiFectamine TM 293 Reagent 4-5 times, then ExpiFectamine TM 293 Mix Reagent and Opti-MENTM I Ruduced Serum Medium, gently blow and invert 2-3 times, and let stand at room temperature for 5 minutes;

[0053] 5) Mix the solutions from steps 3) and 4), gently blow and invert 2-3 times to mix thoroughly;

[0054] 6) Place the solution from step 5) at room temperature for 10-20 minutes;

[0055] 7) Slowly aspirate the mixture into the cell culture medium and gently shake the Erlenmeyer flask;

[0056] 8) Incubate in 8% CO2 at 37°C on a shaker for 5-7 days;

[0057] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer1 and ExpiFectamine TM 293 Transfection Enhancer2 (Note: Mix before use), gently shake to mix, and continue culturing.

[0058] III. Purification of Monoclonal Antibodies

[0059] The antibody was purified from the supernatant using HiTrap rProtein A FF affinity chromatography according to the manufacturer's instructions. Purity was determined by SDS-PAGE gel chromatography, and concentration was determined using the Bradford method. The purified antibody was stored at -20°C.

[0060] Example 4. Immunohistochemical tissue microarray staining and identification

[0061] I. Chip fabrication process

[0062] Each sample was first stained with hematoxylin and eosin (HE) to determine the tumor location. The tumor target sites were circled, and holes were prepared for drilling. When preparing the blank recipient paraffin block, a plastic frame was placed on a mold, and melted paraffin (melting point 55–58℃) was poured into the mold. After cooling to room temperature, the mold was placed in a -20℃ freezer for 6 minutes, and then the paraffin block was removed from the mold. Using a tissue sampling instrument, a 1mm diameter sampling needle was used to drill holes 3–4 mm deep in the recipient paraffin block. Another 1mm diameter needle was used to drill holes at the marked locations in the paraffin block to collect tissue cores, with the cores approximately 0.1mm shallower than the holes in the recipient paraffin block. The collected tissue cores were directly inserted into the holes in the recipient paraffin block or carefully picked up with tweezers and inserted. This process was repeated until all sample sites were prepared. Finally, all tissue cores were flattened with a glass slide to make the tissue chip paraffin block flat and smooth. The prepared tissue chip wax block is placed into a wax block mold and placed in a 60℃ oven for 15 minutes to fuse the tissue core with the recipient wax block. The mold is then gently removed from the oven, and the semi-molten paraffin is allowed to cool at room temperature for about 30 minutes. It is then frozen at -20℃ for 6 minutes. The tissue chip wax block is then removed from the mold and sectioned or stored at 4℃ for later use. After trimming, serial sections are prepared with a thickness of 3μm. The serial sections are then floated in cool water to allow them to spread naturally. Separate sections are then transferred to 45℃ warm water for 30 seconds. The sections are mounted on poly-L-lysine-treated slides. The prepared tissue chip is then baked in a 65℃ oven for 2 hours, removed, cooled to room temperature, and stored at -4℃.

[0063] II. IHC Staining and Analysis

[0064] Dewaxing was performed three times with xylene for 6 minutes each time, followed by hydration in a gradient of 100%, 100%, 95%, and 85% ethanol for 3 minutes each time, and finally rinsing with tap water. Antigen retrieval was then performed, followed by placing the slides in a humidified chamber and rinsing with PBS for 3 x 3 minutes. Incubation with 3% H2O2 for 10 minutes was then performed, followed by rinsing with PBS for 3 x 3 minutes. The slides were then dehydrated, and a suitable diluted primary antibody (the initial dilution should be designed based on the antibody concentration) was added. Incubation was performed at room temperature (25°C) for 1 hour, followed by rinsing with PBS for 3 x 3 minutes. Secondary antibody was added and incubated at room temperature for 15-30 minutes, followed by rinsing with PBS for 3 x 3 minutes. The PBS was discarded, and the slides were developed with freshly prepared DAB chromogenic solution for 3-10 minutes. Hematoxylin was counterstained for 25 seconds, followed by PBS blue staining for 30 seconds. Dehydration was performed sequentially with an ethanol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes). Finally, the slides were cleared with xylene for 3 minutes and mounted with neutral resin.

[0065] Immunohistochemical staining results are categorized as positive or negative. Positive expression must occur at a specific antigenic site on the cell or tissue to be considered positive. When tissue staining distribution is clear and cell localization is accurate, the staining results are further subdivided based on differences in staining intensity, as follows:

[0066] 1. The sample is weakly positive; it is marked as "+";

[0067] 2. The sample is moderately positive; marked as "++";

[0068] 3. The sample is highly positive; it is marked as "+++".

[0069] 4. If the sample is negative, mark it as "-".

[0070] III. Data Statistics

[0071] 1. Tumor tissue microarray detection results:

[0072] The antibody FRα (MXR066) of this invention and the commercially available antibody FRα (BN3.2) were used to simultaneously detect and compare the results in 26 cases of ovarian serous carcinoma. The immunohistochemical results of FRα were statistically analyzed. The entire trial was conducted using a double-blind design, and the statistical results are shown in the table below:

[0073]

[0074] The results showed that the rabbit monoclonal antibody FRα (MXR066) provided accurate staining localization, clear staining without nonspecific staining, and a clean background. In immunohistochemical detection, the positive rate was comparable to that of commercially available antibodies, with 12 cases showing higher positive intensities for the rabbit FRα antibody compared to commercially available antibodies, indicating that the sensitivity of the rabbit monoclonal antibody FRα (MXR066) is higher than that of commercially available antibodies.

[0075] Figure 1 Comparison of immunohistochemical staining results of one case of ovarian serous carcinoma (left: rabbit monoclonal antibody FRα of this invention, right: commercially available FRα).

[0076] 2. Results of microarray analysis of normal tissue:

[0077] The normal tissue microarray includes 30 types of normal tissue samples, primarily selected from fresh, promptly fixed surgical specimens; each tissue type includes three different case samples. The 30 normal tissue types include: brain, heart, cerebellum, esophagus, adrenal glands, stomach, ovary, small intestine, pancreas, colorectal region, parathyroid glands, liver, pituitary gland, salivary glands, testes, kidneys, thyroid gland, prostate, breast, uterus, spleen, bladder, tonsils, skeletal muscle, thymus (in young children), skin, bone marrow, peripheral nerves, lungs, and mesothelial cells.

[0078] The rabbit monoclonal antibody FRα (MXR066) and a commercially available FRα antibody were simultaneously detected on a normal tissue microarray. The positive and negative test results were consistent, indicating that the specificity of this antibody in normal tissues is comparable to that of the commercially available antibody. Figure 2 Comparison of immunohistochemical staining results of fallopian tube tissue (left: rabbit monoclonal antibody FRα of this invention, right: commercially available FRα).

[0079] Finally, it should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.

Claims

1. A monoclonal antibody against FRα protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a rabbit monoclonal antibody with clone number MXR066.

3. A method for preparing a monoclonal antibody against FRα protein, characterized in that, The antigen used to immunize rabbits is a recombinant protein, which is recombinantly expressed by Escherichia coli.

4. The preparation method according to claim 3, characterized in that, The recombinant protein contains an FRα protein fragment and an HIS protein tag.

5. The preparation method according to claim 3, characterized in that, The FRα protein fragment has the amino acid sequence shown in SEQ ID NO.

3.

6. The preparation method according to claim 3, characterized in that, The plasmid vector used in the recombination process was PCMV3.

7. An immunoassay reagent for FRα protein, characterized in that, The immunoassay reagent contains the anti-FRα protein monoclonal antibody as its active ingredient, as described in claim 1.

8. The immunoassay reagent according to claim 7, characterized in that, The immunoassays include immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

Images